Project summary/abstract: The long-term goal of this research project is to gain an understanding of the broader relationship between phenobarbital-resistant seizures in neonates, the role of KCC2 hypofunction in the emergence of refractory seizures, and the efficacy of novel KCC2 functional enhancers in a mouse model of neonatal ischemic seizures. Refractory neonatal seizures are highly correlated with childhood seizure syndromes and cognitive disabilities. The development of more effective therapies will benefit from a deeper understanding of the pathophysiology and mechanisms of underlying refractoriness and epileptogenesis in animal models. The KCC2 chloride co-transporter is the chief Cl- extruder in central nervous system neurons. Severe impairment in a neurons ability to extrude Cl- reverses the transmembrane Cl- gradient resulting in GABA mediated depolarization instead of hyperpolarization. Excitotoxic insults in neonatal brains are often associated with severe seizure burdens that are commonly refractory to first-line therapeutic interventions with GABA agonists like phenobarbital. Our previous work has shown that ischemia significantly downregulates Cl- co-transporter KCC2 expression but NKCC1 expression which is the Cl- importer remains unaffected with trends of upregulation in post-ischemic brains. Rescuing the pathophysiological hypofunction of KCC2 following ischemic insults is an untested strategy in neonatal brains. Hypothesis: Rescuing KCC2 hypofunction in neonatal ischemia will restore the physiological levels of synaptic inhibition and neuronal network activity. This rescue will prevent the emergence of refractory seizures and successfully reduce seizure burdens with GABA agonists which in turn will be disease modifying in the long-term. Aims: 1.Plot the dynamics of early and acute KCC2 degradation following ischemia and investigate the regulation of intrinsic KCC2 hypofunction during ischemic seizures. 2. Document the KCC2 degradation related depolarization of cortical neurons following ischemia and the effects of a KCC2 agonist on such depolarization in-vitro 3. Rescue refractory ischemic - seizures in-vivo with a novel KCC2 agonist and quantitate effect on long-term co-morbidities. Deliverables: Upon successful completion of this project, we will move closer to understanding the link between the dynamic changes of KCC2 expression during neonatal seizures to the emergence of refractoriness. Impact and Innovation: Understanding the mechanisms by which the immature brain is transformed with repeated seizures in the neonatal period will help guide evidence-based strategies into treatments for intractable seizures that are often associated with severe long-term co-morbidities in children.